Tributylethylammonium ethyl sulphate

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2017 - 03 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Samples were taken from all concentrations and the control according to the schedule below:
Frequency: at t=0 h and at t=48 h
Volume: 2.0 mL
Storage: Samples were stored in a freezer (≤ 15°C) until analysis.

At the end of the exposure period, the replicates were pooled at each concentration before sampling.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Direct application to the test medium: preparation of test solutions for the range-finding test and the final test started with the highest concentration of 100 mg/L (37 mg anion/L). Since the test item was completely soluble in test medium, no other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium.
- Controls: Test medium without test item or other additives.
- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia
- Age at study initiation: <24 hours
- Source: in-house laboratory culture with a known history, at least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions. The daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age of parental stock: >2 weeks
- Feeding during test: no

BREEDING
- Breeding method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. Cultures (maximum age of 4 weeks) were renewed after 7 days of cultivation by renewing the medium twice a week.
- Feeding: daily, a suspension of fresh water algae.
- Health during breeding period (any mortality observed): no mortality observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

180 mg CaCO3/L

Test temperature:

20 - 21°C

pH:

Start: 7.9-8.0
End: 8.2-8.3

Dissolved oxygen:

Start: 8.9 mg O2/L
End: 8.8-9.1 mg O2/L

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg test item/L (corresponding to 3.7, 6.6, 12, 21 and 37 mg anion/L)
Measured concentrations at t=0: 10, 18.5, 32.0, 57 and 100 mg test item/L based on the anion concentration and 10, 19.0, 33.4, 58.1 and 106 mg test item/L based on cation concentration
Concentrations at t=72 were 98-102% of initial test solutions based on the anion concentration and 96-101% of initial based on the cation concentration.
Based on these results the effect concentrations were based on the nominal concentrations mg test item/L calculated from the nominal concentrations of anion.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass vessels, open, fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water purified by Reverse Osmosis
- Culture medium different from test medium: yes, M7 medium
- Intervals of water quality measurement: at t=0 h and at t=48 h (dissolved oxygen and pH measurements)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours daily

EFFECT PARAMETERS MEASURED: immobility at t=24 and t=48 hours

COMBINED LIMIT/RANGE-FINDING STUDY
- Test concentrations: 0.10, 1.0, 10 and 100 mg/L (0.037, 0.37, 3.7 and 37 mg anion/L)
- Results used to determine the conditions for the definitive study: yes, in the highest concentration 60% of the daphnids became immobilized after 24 hours and 100% was immobilized after 48 hours. The expected EC50 was between 10 and 100 mg test item/L.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (June 2017)

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

34 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 30-40 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

nominal

Conc. based on:

other: anion

Basis for effect:

mobility

Remarks on result:

other: 95% CI: 11-15 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

21 mg/L

Nominal / measured:

nominal

Conc. based on:

other: cation

Basis for effect:

mobility

Remarks on result:

other: cation concentration calculated

Details on results:

- Any abnormalities observed: no
- No immobilisation was observed in the control and the lowest concentration tested. A dose-related increase of immobilisation was observed at the end of the test.Microscopic observations at t=24 h (in one replicate vessel) showed that there was no test item attached to the daphnids exposed to the highest concentration. It was therefore concluded that the observed immobility was not due to mechanical obstruction.
- Actual exposure concentrations were confirmed by analysis at the start and at the end of the test. However, 3 out of 5 concentrations were calculated by extrapolation of the calibration curve of the anion. Since cation results where acceptable (the same results with the curve with broader range) and not extrapolated, all values for anion concentrations are accepted without additional analysis.
- Analysis showed that measured concentrations of the anion were 98-102% of nominal concentrations after the 72 hour exposure period. Effect parameters were expressed in terms of analytically confirmed nominal concentrations of the anion and calculated to mg test item/L.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested: 0.10, 0.18, 0.32, 0.56, 1.0 and 1.8 mg/L
- Relevant effect levels: 48h-EC50: 0.59 mg/L, 95% CI: 0.52-0.66 mg/L
- Other: the results where within the expected range according to the historical data (>0.3 and <1.0 mg/L)

Reported statistics and error estimates:

The 24 and 48h-EC50-value was calculated from the probits of the percentages of affected daphnids and the logarithms of the corresponding nominal concentrations of the anion using the maximum likelihood estimation method.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1 Measured concentrations (anion) versus nominal concentrations

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	< 1	n.a.
	10	10^*,#	101
	18	18.5	103
	32	32.0	100
	56	57^*,#	101
	100	100^*,#	100
48	0	< 1	n.a.	n.a.
	10	10^*,#	100	99
	18	18.4	102	99
	32	32.7	102	102
	56	57^*,#	102	101
	100	98^*,#	98	98

^*     Estimated value, calculated by extrapolation of the calibration curve.

^#     Since cation results where acceptable (the same results with the curve with broader range) and not extrapolated, anion results are accepted without additional analysis.

n.a. Not applicable.

Table 1 Measured concentrations (cation) versus nominal concentrations

Time of sampling [hours]	Concentration [mg/L]		Relative to nominal [%]	Relative to initial [%]
	Nominal	Analyzed
0	0	< 0.1	n.a.
	10	10.0	100
	18	19.0	106
	32	33.4	104
	56	58.1	104
	100	106	106
48	0	< 0.1	n.a.	n.a.
	10	9.97	100	99
	18	19.0	105	100
	32	33.6	105	101
	56	58.7	105	101
	100	102	102	96

n.a.Not applicable.

Table 2 Number of introduced daphnids and incidence of immobility in the final test

Time (h)	Replicate	Nominal concentration TBEAES DRY (mg anion/L)
		Control	3.7 (10)	6.6 (18)	12 (32)	21 (56)	37 (100)
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	1
	B	0	0	0	0	1	2^#
	C	0	0	0	0	0	1
	D	0	0	0	0	1	1
	Total immobilised	0	0	0	0	2	5
	Effect %	0	0	0	0	10	25
48	A	0	0	0	1	4	5
	B	0	0	0	2	5	5
	C	0	0	0	2	5	5
	D	0	0	1	2	5	5
	Total immobilised	0	0	1	7	19	20
	Effect %	0	0	5	35	95	100

() – between brackets, the concentration of the test item are given (mg/L),^#Microscopic observation revealed no test item attached to the daphnids

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Conclusions:

The 48h-EC50 for Daphnia magna exposed to TBEAES DRY was 34 mg/L based on the nominal concentration of the test item.

Executive summary:

In a short-term toxicity test, performed according to OECD guideline 202 and GLP principles, TBEAES DRY was assessed for its potential toxicity towards Daphnia magna. The test item concentration was corrected for the concentration of anion with a correction factor of 0.368.

Test solutions were prepared by adding the test item directly to the test medium and shaking the solutions vigorously for complete dissolution of the test item. A combined limit/range-finding test was performed with test concentrations of 0.1, 1.0, 10 and 100 mg/L (0.037, 0.37, 3.7 and 37 mg anion/L).

In the final test twenty daphnids were exposed to a blank control and test item concentrations of 10, 18, 32, 56 and 100 mg/L (3.7, 6.6, 12, 21 and 37 mg anion/L) under static conditions for 48 hours. Samples were taken from the control and all concentrations at the start and at the end of the test for confirmation of exposure concentrations. The analysis was based on the concentration of anion and cation in the test solutions. Test item concentrations were calculated from the anion and cation concentrations and results for the two were comparable. Measured concentrations of the anion were 98-102% of nominal concentrations after the 72 hour exposure period and 96 -101% for the cation. Effect parameters were expressed in terms of analytically confirmed nominal concentrations of the anion and calculated to mg test item/L.

Incidence of immobility was recorded at t=24 and t=48. The 48h-EC50 for Daphnia magna exposed to TBEAES DRY was determined to be 34 mg test item/L (13 mg anion/L). The acceptability criteria were met and the study was considered to be valid.

Description of key information

In a short-term toxicity test, performed according to OECD guideline 202 and GLP principles, TBEAES DRY was assessed for its potential toxicity towards Daphnia magna. The test item concentration was corrected for the concentration of anion with a correction factor of 0.368. Test solutions were prepared by adding the test item directly to the test medium and shaking the solutions vigorously for complete dissolution of the test item. A combined limit/range-finding test was performed with test concentrations of 0.1, 1.0, 10 and 100 mg/L (0.037, 0.37, 3.7 and 37 mg anion/L). In the final test twenty daphnids were exposed to a blank control and test item concentrations of 10, 18, 32, 56 and 100 mg/L (3.7, 6.6, 12, 21 and 37 mg anion/L) under static conditions for 48 hours. Samples were taken from the control and all concentrations at the start and at the end of the test for confirmation of exposure concentrations. The analysis was based on the concentration of anion and cation in the test solutions. Test item concentrations were calculated from the anion and cation concentrations and results for the two were comparable. Measured concentrations of the anion were 98-102% of nominal concentrations after the 72 hour exposure period and 96 -101% for the cation. Effect parameters were expressed in terms of analytically confirmed nominal concentrations of the anion and calculated to mg test item/L. Incidence of immobility was recorded at t=24 and t=48. The 48h-EC50 for Daphnia magna exposed to TBEAES DRY was determined to be 34 mg test item/L (13 mg anion/L). The acceptability criteria were met and the study was considered to be valid.